Cellular and molecular mechanisms involved in T cell-mediated autoimmunity against immunologically privileged retinal antigens are being studied. The questions are aimed at elucidating the natural development and maintenance of self-tolerance to retinal antigens, and at defining the processes that lead to their pathological breakdown. The goal is to use this knowledge for designing novel and rational strategies for immunotherapy. The experimental approaches utilize the model of experimental autoimmune uveoretinitis (EAU), which resembles immune-mediated uveitic diseases in humans that can lead to blindness. EAU is induced in mice and rats by immunization with retinal antigens such as IRBP, Arrestin (S-Ag), or their component peptide epitopes, or by infusion of cultured lymphocytes that recognize these antigens. The mechanisms controlling disease susceptibility and pathogenesis are being defined at the genetic, developmental, and immunological levels. Novel approaches to disease regulation are devised based on these findings. Natural tolerance to IRBP is being investigated by using mice that differ in IRBP expression. 1) IRBP-transgenic (TG) mice that express the N-terminal half of IRBP extraocularly under control of the MHC class II promoter. 2) IRBP knockout (KO) mice that have no detectable expression of IRBP and 3) wild type (WT) mice expressing IRBP in the eye and pineal. Using these mice, we found that thymic selection plays a major role in tuning the threshold of susceptibility to ocular autoimmunity. The mechanism involves elimination of highly IRBP reactive T cells and release of CD4+CD25+ regulatory T cells by the thymus. Extraocular expression of IRBP can enhance this tolerance and abolishes the ability to develop EAU in a transgene-specific fashion. These results suggested that thymic tolerance has a greater role than hitherto realized in setting the threshold of immune and autoimmune responsiveness to sequestered retinal antigens. In contrast, peripheral tolerance appears to be an underutilized mechanism that may be exploited therapeutically. Based on this conclusion, we are studying therapeutic induction of tolerance in adult mice by two distinct genetic approaches. 1) an immunodominant IRBP fragment is expressed in autologous B cells by ex vivo transduction with a retroviral construct. Infusion of these cells into genetically compatible recipients results in a profound and long-term tolerance that can both prevent and reverse EAU. 2) IV or IM vaccination with a DNA plasmid encoding an IRBP fragment. This method is efficient in preventing disease, but less so in reversing disease elicited by already primed T cells. The mechanism of protection in both DNA vaccination and B cell therapy appears to involve inactivation of high-affinity Ag specific T cells. This conclusion is based on data showing that although immunological hyporesponsiveness is induced, protection is not transferable and does not involve the two major known apoptosis pathways involving Fas/FasL (death receptor driven) or Bcl-2 regulated (mitochondrial). Transforming growth factor beta (TGF-b) is an immunoinhibitory factor found in aqueous humor of the eye and known to suppress T cell responses. We found that although it can inhibit the activation and function of immature (primary culture) uveitogenic effector T cells, polarized uveitogenic T cells (long term line) are insensitive to TGF-b and may even be enhanced. The mechanism involves direct effects on the T cell as well as indirect effects through the antigen-presenting cell. In chronic uveitis polarized effector T cells may help drive the disease. This may explain persistence of uveitis in the face of the high TGF-b levels found in the eye. Innate immune responses to microbes, that create a pro-inflammatory environment, are thought to play a role as environmental triggers of autoimmunity. For this reason we have made efforts to delineate the role of adjuvant effects in EAU. Pertussis toxin (PT) enhances autoimmune disease in experimental models. PT administered at the time of immunization enhances the Th1 response, which underlies EAU pathology, but can also inhibit EAU if given later, by preventing effector cell migration through blockade of chemokine signaling. We have now resolved these opposing effects of PT. Whereas the disease-inhibitory ability resides in the A subunit, the disease-enhancing activity resides primarily in the B subunit. In contrast to PT, cholera toxin, which shares structural, but not functional, homology with PT, appears to inhibit EAU through induction of immune deviation. Using gene knockout mice developed at Osaka University in Japan, we are studying the TOLL receptor dependence of adjuvant effects in EAU. Preliminary data using TLR-deficient mice indicate that different TLRs can have redundant functions in promoting EAU induction. In collaboration with the Oppenheim group at NCI we demonstrated that lymphocytes and immature dendritic cells exhibit chemotactic responses to the retinal antigens S-Ag and IRBP. The chemokine receptors CXCR5 and CXCR3 mediated the chemotactic effect of IRBP, while only CXCR3 was required for the S-Ag signal. We hypothesize that these responses may underlie attraction of immunocytes to sites of tissue damage and may thus facilitate processes of tissue repair and regeneration. In conjunction with adjuvant effects such as a concomitant infection, autoimmune-prone individuals having inadequate immunological control mechanisms, such responses may lead to development of an adaptive response of the Th1 phenotype, leading to autoimmune tissue pathology. These studies will help to understand disease modulation by adjuvant effects. EAU, similarly to uveitis in humans, is genetically controlled by MHC as well as non-MHC genes. We study genetic control of EAU using two approaches: 1) defining genetic markers associated with susceptibility in genetically defined rodents. We identified 3 chromosomal regions affecting EAU susceptibility, that are now being isolated in lines of consomic rats by marker-assisted selection. Genes differentially expressed by susceptible and resistant strains are being identified using DNA microarrays. These studies will permit closer identification of the genes and pathways determining susceptibility or resistance to EAU. 2) We have developed a "humanized" model of EAU using HLA-transgenic mice in which murine MHC genes have been replaced by human MHC genes. Interestingly, one of the "humanized" strains is developing strong EAU when immunized with S-Ag, which induces little or no uveitis in WT mice, but elicits responses in humans, and recognize an epitope that is recognized by uveitis patients. These results validate EAU as a model of human uveitis.